NO‐degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR‐1a gene expression in Mhb1‐overproducing tobacco plants
NO‐degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR‐1a gene expression in Mhb1‐overproducing tobacco plants
复制标题
DOI：
10.1016/j.febslet.2004.06.055
复制
发表时间：
2004-07
期刊：
影响因子：
3.5
通讯作者：
Csaba Seregélyes;A. Igamberdiev;A. Maassen;J. Hennig;D. Dudits;R. Hill
中科院分区：
文献类型：
--
作者：
Csaba Seregélyes;A. Igamberdiev;A. Maassen;J. Hennig;D. Dudits;R. Hill
过量产生苜蓿 1 类血红蛋白的烟草植物（HOT 植物）已被证明可以减少坏死症状的发生。在这里，我们表明这种改变的致病反应与转基因植物中受一氧化氮（NO）影响的发病机制相关（PR-1a）转录物积累的显着增加有关。还发现 HOT 转基因幼苗的匀浆比非转化幼苗具有更高的 NO 清除活性。重组苜蓿 1 类血红蛋白的 NO 清除特性已被检测。重组 Mhb1 (rMhb1) 在细菌中产生，并使用聚乙二醇 (10-25%) 分级、DEAE-Sephacel 色谱柱和 Phenyl Superose 柱进行纯化。最终纯化步骤后，获得的制剂接近均质，通过尺寸排阻色谱测定分子量为 44 kDa，通过 SDS-PAGE 测定分子量为 23 kDa，表明 rMhb1 是二聚体。该蛋白以 NAD(P)H 作为辅助因子参与 NO 降解活性。离子交换柱后，需要添加 FAD 以发挥最大的 NO 降解活性。 rMhb1 的 NAD(P)H 依赖性 NO 清除活性与大麦血红蛋白相似，支持这样的结论：单子叶植物和双子叶植物 1 类血红蛋白均可通过清除缺氧、病原体攻击和其他应激过程中形成的 NO 来影响细胞 NO 水平。
Tobacco plants overproducing alfalfa class 1 hemoglobin (HOT plants) have been shown to have reduced necrotic symptom development. Here, we show that this altered pathogenic response is linked to a significant increase in the nitric oxide (NO)-affected pathogenesis-related (PR-1a) transcript accumulation in the transgenic plants. Homogenates of HOT transgenic seedlings were also found to have higher NO-scavenging activity than non-transformed ones. The NO-scavenging properties of recombinant alfalfa class1 hemoglobin have been examined. Recombinant Mhb1 (rMhb1) was produced in bacteria and purified using polyethylene glycol (10–25%) fractionation, chromatography on DEAE–Sephacel, and Phenyl Superose columns. After the final purification step, the obtained preparations were near homogeneous and had a molecular weight of 44 kDa determined by size-exclusion chromatography and 23 kDa by SDS–PAGE, indicating that rMhb1 is a dimer. The protein participated in NO-degradation activity with NAD(P)H as a cofactor. After ion-exchange columns, addition of FAD was necessary for exhibiting maximal NO-degradation activity. The NAD(P)H-dependent NO-scavenging activity of rMhb1, which is similar to that of barley hemoglobin, supports a conclusion that both monocot and dicot class 1 hemoglobins can affect cellular NO levels by scavenging NO formed during hypoxia, pathogen attack and other stresses.